Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may amplify the glucose-induced Ca2+ entry into islet ?-cells that triggers insulin secretion. Ca2+ loss from ?-cell intracellular compartments has been proposed to induce both Ca2+ entry and events dependent on arachidonate metabolism. We examine here effects of inducing Ca2+ loss from intracellular sequestration sites with ionophore A23187 and thapsigargin on arachidonate hydrolysis from islet phospholipids. A23187 induces a decline in islet arachidonate-containing phospholipids and release of nonesterified arachidonate. A23187-induced arachidonate release is of similar magnitude when islets are stimulated in Ca2+-replete or in Ca2+-free media or when islets loaded with the intracellular Ca2+ chelator BAPTA are stimulated in Ca2+-free medium, a condition in which A23187 induces no rise in ?-cell cytosolic [Ca2+]. Thapsigargin also induces i sletarachidonate release under these conditions. A23187-or thapsigargin-induced arachidonate release is prevented by a bromoenol lactone (BEL) inhibitor of a ?-cell phospholipase A2 (iPLA2) which does not require Ca2+ for catalytic activity and which is negatively modulated by and physically interacts with calmodulin by Ca2+-dependent mechanisms. Agents which cause Ca2+ loss from islet intracellular compartments thus induce arachidonate hydrolysis from phospholipids by a BEL-sensitive mechanism that does not require a rise in cytosolic [Ca2+], and a BEL-sensitive enzyme like iPLA2 or a related membranous activity may participate in sensing Ca2+ compartment content.